Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

Abstract

Non-coding RNAs play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). The aim of this study was to characterize the therapeutic role of melatonin as well as the underlying molecular mechanism (its effects on the expression of H19 and its downstream signaling pathways) in the treatment of PAH. Real-time PCR and western blot analysis were performed to evaluate the expression of H19, miR-200a, miR-675, insulin-like growth factor-1 receptor (IGF1R), and programmed cell death 4 (PDCD4). The value of systolic pulmonary artery pressure (SPAP) and the ratio of medial thickening in the monocrotaline (MCT) group were increased, whereas the melatonin treatment could decrease these values to some extent. The weights of RV (right ventricle), LV (left ventricle) + IVS (interventricular septal), and RV/(LV + IVS) in the MCT group were much higher than those in the MCT + melatonin and control groups. In addition, the expression of H19, miR-675, IGF1R mRNA, and IGF1R protein in the MCT group was the highest, whereas their expression in the control group was the lowest. The expression of miR-200, PDCD4 mRNA, and PDCD4 protein in the MCT group was the lowest, whereas their expression in the control group was the highest. Furthermore, H19 directly suppressed the expression of miR-200a, whereas miR-675-3p and miR-200a directly inhibited the expression of IGF1R and PDCD4, respectively. Finally, melatonin treatment inhibited cell proliferation; upregulated the expression of H19, miR-675-3p, and PDCD4; and downregulated the expression of miR-200a and IGF1R. This study demonstrated the role of H19-miR-675-3p-IGF1R- and H19-miR-200a-PDCD4-signaling pathways in the melatonin treatment of PAH.

Keywords: H19; apoptosis; melatonin; miR-200a; miR-675; pulmonary arterial hypertension.

Figure 1

Changes in SPAP and Organ Weights after MCT Treatment in PAH Rats (A) SPAP in the MCT group was much higher than in the MCT plus melatonin group, and both of them were much higher than the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (B) Weight of RV in the MCT group was much higher than in the MCT plus melatonin and control groups, and both of them were higher than that of the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (C) The MCT group exhibited a lower weight of LV + IVS compared with the MCT plus melatonin group, and the MCT plus melatonin group showed a lower weight of LV + IVS than the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (D) The MCT group exhibited a higher RV/(LV + IVS) compared with the MCT plus melatonin group, and the MCT plus melatonin group showed a higher RV/(LV + IVS) than the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (E) Ratio of medial thickening in the MCT group was much higher than in the MCT plus melatonin group, and both of them were much higher than the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group).

Figure 2

Differential Levels of H19, miR-675, miR-200a, IGF1R, and PDCD4 after MCT Treatment in PAH Rats (A) The H19 level in the MCT plus melatonin group was much higher than in the MCT group, while it was even higher in the control group than in the MCT plus melatonin group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (B) The miR-200a level in the MCT plus melatonin group was much higher than in the control group, while it was even higher in the MCT group than in the MCT plus melatonin group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (C) The miR-675-3p level in the MCT group was much lower than in the MCT plus melatonin group, and both of them were much lower than in the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (D) The IGF1R mRNA level in the MCT group was much higher than in the MCT plus melatonin group, and both of them were much higher than in the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (E) The MCT plus melatonin group displayed a lower level of PDCD4 mRNA than did the control group, while the MCT group exhibited an even lower level of PDCD4 mRNA than did the MCT plus melatonin group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group). (F) The IGF1R protein level in the MCT group was much higher than in the MCT plus melatonin group, and both of them were much higher than in the control group. The PDCD4 protein level in the MCT group was much lower than in the MCT plus melatonin group, and both of them were much lower than in the control group (*p < 0.05 as compared with the control group, #p < 0.05 as compared with the MCT group).

Figure 3

Immunohistochemistry for IGF1R According to the IHC result, the IGF1R protein level in the MCT group was much higher than in the MCT plus melatonin group, and both of them were much higher than in the control group (scale bars, 10 μm).

Figure 4

Immunohistochemistry for PDCD4 According to the IHC result, the PDCD4 protein level in the MCT group was much lower than in the MCT plus melatonin group, and both of them were much lower than in the control group (scale bars, 10 μm).

Figure 5

H19 Directly Regulated miR-200a (A) Comparison of the sequences of H19 and mutant with replacement of binding site against the sequence of miR-200a. (B) miR-200a mimic inhibited luciferase activity of wild-type H19, but not that of mutant H19 in hPASMCs (*p < 0.05 as compared with the control group). (C) miR-200a mimic reduced luciferase activity of wild-type H19 compared with scramble control, and luciferase activity of mutant H19 showed no obvious difference with scramble control in rPASMCs (*p < 0.05 as compared with the control group).

Figure 6

miR-675-3p and miR-200a Directly Targeted IGF1R and PDCD4, Respectively (A) The sequence comparison between mature miR-675-3p and wild-type as well as mutant IGF1R 3′ UTR. (B) Luciferase activity of wild-type IGF1R 3′ UTR, but not that of mutant IGF1R 3′ UTR, in hPASMCs transfected with miR-675-3p mimic was much lower than scramble control (*p < 0.05 as compared with the control group). (C) Luciferase activity of wild-type IGF1R 3′ UTR, but not that of mutant IGF1R 3′ UTR, in rPASMCs transfected with miR-675 mimic was much lower than scramble control (*p < 0.05 as compared with the control group). (D) The sequence comparison between mature miR-200a and wild-type as well as mutant PDCD4 3′ UTR. (E) Luciferase activity of wild-type PDCD4 3′ UTR, but not that of mutant PDCD4 3′ UTR, in hPASMCs transfected with miR-675 mimic was downregulated compared with scramble control (*p < 0.05 as compared with the control group). (F) Transfecting with miR-200a mimic reduced luciferase activity of wild-type PDCD4 3′ UTR, but not that of mutant PDCD4 3′ UTR, in rPASMCs (*p < 0.05 as compared with the control group).

Figure 7

Effect of Melatonin on Cell Proliferation and H19, miR-675-3p, miR-200a, IGF1R, and PDCD4 Levels in hPASMCs (A) Melatonin inhibited cell viability of hPASMCs in a dose-dependent manner (*p < 0.05 as compared with the control group). (B) Melatonin inhibited cell viability of hPASMCs in a dose-dependent fashion. (C) Treating with melatonin dose-dependently upregulated H19 expression (*p < 0.05 as compared with the control group). (D) Treating with melatonin dose-dependently enhanced miR-675-3p expression (*p < 0.05 as compared with the control group). (E) miR-200a level was dose-dependently reduced following treatment with melatonin (*p < 0.05 as compared with the control group). (F) IGF1R mRNA level was dose-dependently suppressed after the administration of melatonin (*p < 0.05 as compared with the control group). (G) PDCD4 mRNA level was dose-dependently upregulated subsequent to treatment with melatonin (*p < 0.05 as compared with the control group). (H) IGF1R protein level was dose-dependently suppressed while PDCD4 protein expression was dose-dependently enhanced following treatment with melatonin.

Figure 8

Effect of Melatonin on Cell Proliferation and H19, miR-675, miR-200a, IGF1R, and PDCD4 Levels in rPASMCs (A) Melatonin inhibited cell viability of rPASMCs in a dose-dependent manner (*p < 0.05 as compared with the control group). (B) Melatonin inhibited cell viability of rPASMCs in a dose-dependent fashion. (C) Treating with melatonin dose-dependently upregulated H19 expression (*p < 0.05 as compared with the control group). (D) Treating with melatonin dose-dependently enhanced miR-675-3p expression (*p < 0.05 as compared with the control group). (E) miR-200a level was dose-dependently reduced following treatment with melatonin (*p < 0.05 as compared with the control group). (F) IGF1R mRNA level was dose-dependently suppressed after the administration of melatonin (*p < 0.05 as compared with the control group). (G) PDCD4 mRNA level was dose-dependently upregulated subsequent to treatment with melatonin (*p < 0.05 as compared with the control group). (H) IGF1R protein level was dose-dependently suppressed while PDCD4 protein expression was dose-dependently enhanced following treatment with melatonin.

Figure 9

Two Signaling Pathways Are Shown Dual signaling pathways (H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4) were involved in the mechanisms underlying the therapeutic effect of melatonin in the treatment of PAH by promoting the apoptosis of PASMCs.